| The hepatitis B virus (HBV) is a widespread human pathogen and chronic HBV infection is a major risk factor for hepatocellular carcinoma (HCC). However, its molecular mechanism has been not very clear.MicroRNA plays important roles in the regulation of interaction of between host cell and virus. The way of regulation by microRNA to virus has two major aspects:on one hand, microRNA directly interacts with the gene of virus or host to assist the replication of the virus. on the other hand, microRNA, which indirectly interacts with the gene of virus or host through inducing mRNA degradation or inhibiting protein translation mechanisms, participates in virus infection, resistance and escape in host cell.Presently, the research of interaction between microRNA and HBV has become more and more. The way of regulation is mainly direct or indirect. After the HBV infection, viruses may therefore gain an advantage by reshaping the cellular miRNA composition. however, the mechanism is not clear. Current some research show that the expression pattern of microRNA has changed after HBV infection. It was speculated that HBV may activate specific cellular transcription factors to repress or increase miRNA transcription or interact with specific cellular factors to interfere with miRNA maturation. This paper has two main results:Firstly, we discovered that miR15a could inhibit HBV replication through directly targeting HBV genome. Firstly, we used RNAi method to detect the effect of micro RNA system on HBV infection. Koncking down Dicer and Ago2, which were important elements of RISC complex, enhanced HBsAg and HBeAg levels as well as viral DNA. At the same time, we selected a panel of micro RNAs previously implicated in inhibiting HBV infections and quantified their expression in response to HBV replication by real time PCR.We found the profile of microRNA has changed after HBV infected into HepG2cell. for example, the expression of miR-15a, miR-16and miR-let-7a were down regulated, however the expression of miR-602, miR-210were up regulated. We found the potential targeting sites of miRNAs by using the soft of RNA22and targetscan. We tested their effects on the luciferase reporters containing the predicted target sequences from HBp and HBx. We found that miR-15a was able to suppress the activity of the HBp luciferase reporter, the mutant miR-15a lacked such effect, and conversely, the miR15a inhibitor enhanced the activity of the luciferase reporter. A similar set of data was also obtained on the luciferase reporter containing the predicated miR-15a targeting site in the HBx transcript.Secondly, HBx mRNA could down regulate the expression of miR-15a/16in the form of protein or RNA. First of all, we made the plasmids, which could express HBx protein or only HBx mRNA. Infected the plamid into HepG2cell, we found microRNA's expression has changed. The result was same with infected pch9-3091into HepG2cell. To continue to find the mechanism, which was how could HBx regulate the expression of miRNAs, we discovered HBx in RNA level could down regulate the expression of miR-15a/16. The data showed that the effect of HBx RNA has abolished when we made HBx RNA mutant unit and transfected then into cells. At last, we suggested that the mutant sites in HBX sequence was important for the mechanism and they could interact with each other directly by the way of base pair.This research can deepen understanding of the mechanism of the interaction between the host miRNA and HBV and provide some new ideas and methods of prevention and treatment of HBV. |
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